To better understand the role of GCK and GCKR in vivo, the murine Gck and Gckr genes have been isolated. Both Gck-/- and Gckr -/- mice have been created and backcrossed on to a C57Bl/6 background used to generate double knock-out (KO) mice. The mutations did not affect mouse development as the Gck, Gckr, and double KO mice are born with normal Mendelian frequencies. We have nearly completed the analysis of these mice and are now focused on the double KO mice as they exhibit the most severe phenotype. B lymphocyte recirculation through lymph nodes requires crossing endothelial barriers and chemoattractant-triggered cell migration. We have shown how lymph node anatomy and chemoattractant receptor signaling organize B lymphocyte trafficking through lymph nodes. Blood borne B cells predominately used CCR7 signaling to adhere to high endothelial venules (HEVs). New B cell emigrants slowly transited the HEV perivenule space, and thereafter localized nearby, avoiding the follicle. Eventually the newly arrived B cells entered the basal portion of the lymph node follicle gradually populating it. During their lymph node residency, B cells reacquired their S1P1 receptors and markedly attenuated their sensitivity to chemokines. The B cells exited the LN follicle by entering the cortical lymphatics or returning to the paracortical cords. Upon entering the lymphatics B cells lost their polarity, downregulated their S1P1 receptors, and subsequently strongly upregulated their sensitivity to chemokines. These results were summarized in a model of homeostatic trafficking of B cells through lymph ndoes. We have also begun a study of how local immunization alters homeostatic lymphocyte trafficking through the draining lymph node. We have shown how immunization induced changes in lymph node architecture along with cell intrinsic factors facilitate and coordinate the trafficking of B cells into and through lymph nodes following immunization. Our studies of chemokine receptor signaling have focused on the proximal elements in the signaling pathway. These receptors predominantly use the heterotrimeric G protein Gi to link to downstream signaling pathways. We have shown Gi alpha proteins regulate and co-localize with F-actin at actin-rich structures, including microspikes or filopodia, lamellipodia, adhesion sites, phagocytic cups, actin comet tails, sub-cortical ruffles and stress fibers. Consistently, reduction of Gi alpha protein expression altered cell morphology, modulated actin filaments and microtubules, and reduced cell migration. That Gi alpha facilitates the interplay of actin and microtubule was further supported by observations that Gi alpha depletion and PTX treatment both altered the dynamics and distribution of myosin X, Rab5, Rac, and the actin bundling protein fascin, which directs myosin X to tips of filopodia. Conversely, CXCL12 treatment induced the translocation of Gi alpha, myosin X, Rab5 and fascin to the plasma membrane, and increased their co-localization at filopodia, periphery ruffle and circular ruffle. As a potential downstream effector in the chemokine receptor signaling pathway, we have examined the functional role of the non-muscle myosin Myo1e, which is significantly enriched in B lymphocytes. We have established Myo1e-/- mice and are studying the consequences of the loss of this protein on B lymphocyte function. Sphingosine-1 Phosphate (S1P) helps mediate lymphocyte egress from lymph nodes, yet significant mechanistic questions remain. We have shown that B lymphocyte egress sites exist adjacent to lymph node follicles. Recent B cell emigrants localize towards follicle centers, while longer-term residents tend towards the cortical sinusoids. Exiting B lymphocytes cross through apparent portals in the lymphatic endothelium. Treatment with the S1P receptor agonist FTY720 empties the cortical sinusoids of lymphocytes, blocks lymphatic endothelial penetration, and displaces B lymphocytes into the T cell zone. S1P3-/- B cells, which lack chemoattractant responses to S1P, transit lymph nodes normally while Gnai2-/- B cells, which have impaired responses to chemokines and S1P, transit more rapidly than do wild type cells. This study identifies a major site of B lymphocyte lymph node egress, shows that FTY720 treatment likely closes cortical lymphatic endothelial portals, and argues against a functional role for S1P chemotaxis in B lymphocyte egress. In a collorbation with Steve Shaw's laboratory (NCI) we have used intravital microscopy to investigate the behavoir of T cells from mice transgenic for ezrin (T567D). This mutation interfers with the dephosphorylation of ezrin that normally occurs with chemokine receptor signaling. We found that T cells from these mice stick normally to high endothelial venules, but have an impairment in transendothelial migration. In addition these T cells have a reduced interstial velocity in the lymph node cortex and an impairment in lymph node egress. This study implicates Ezin dephosphoryaltion as important step for T lymphocyte transmigration and for normal motility. Autophagy delivers cytoplasmic constituents to autophagolysosomes and is linked to both innate andadaptive immunity. Toll-like receptor 4 (TLR4) signaling induces autophagy and recruits Beclin-1, the mammalian homolog of yeast Atg6, to the receptor complex. We found that tumor necrosis factor receptor (TNFR)associated factor 6 (TRAF6)mediated, Lys63 (K63)linked ubiquitination of Beclin-1 is critical for TLR4-triggered autophagy in macrophages. Two TRAF6-binding motifs in Beclin-1 facilitated the binding of TRAF6 and the ubiquitination of Beclin-1. Lys117, which is strategically located in the Bcl-2 homology 3 (BH3) domain of Beclin-1, was a major site for K63-linked ubiquitination. The deubiquitinating enzyme A20 reduced the extent of K63-linked ubiquitination of Beclin-1 and limited the induction of autophagy in response to TLR signaling. Treatment of macrophages with either interferon-g or interleukin-1 also triggered the K63-linked ubiquitination of Beclin-1 and the formation of autophagosomes. These results indicate that the status of K63-linked ubiquitination of Beclin-1 plays a key role in regulating autophagy during inflammatory responses. Inflammasomes are molecular platforms activated by infection or stress that regulate the activity of caspase-1 and the maturation of interleukin 1 beta and IL-18. Suggesting interplay between these pathways, lipopolysaccharide induces inflammasome activation in macrophages genetically deficient in autophagy proteins, but not in wild type macrophages. Here we show that the induction of the AIM2 inflammasome in macrophages triggers autophagosome formation, which results in the engulfment and degradation of inflammasomes. Blocking autophagy potentiated AIM2 inflammasome activity. AIM2 inflammasomes underwent lysine-63 linked ubiquitination allowing the recognition of inflammasomes by the autophagic adaptor p62. These results show that autophagy can limit inflammation by eliminating already formed AIM2 inflammasomes.